Did the exposure of coacervate droplets to rain make them the first stable protocells?

Membraneless coacervate microdroplets have long been proposed as model protocells as they can grow, divide, and concentrate RNA by natural partitioning. However, the rapid exchange of RNA between these compartments, along with their rapid fusion, both within minutes, means that individual droplets would be unable to maintain their separate genetic identities. Hence, Darwinian evolution would not be possible, and the population would be vulnerable to collapse due to the rapid spread of parasitic RNAs. In this study, we show that distilled water, mimicking rain/freshwater, leads to the formation of electrostatic crosslinks on the interface of coacervate droplets that not only suppress droplet fusion indefinitely but also allow the spatiotemporal compartmentalization of RNA on a timescale of days depending on the length and structure of RNA. We suggest that these nonfusing membraneless droplets could potentially act as protocells with the capacity to evolve compartmentalized ribozymes in prebiotic environments.


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Notes S1 to S3 Figs.S1 to S15 Table S1 Legend for movie S1 References Other Supplementary Material for this manuscript includes the following: Movie S1

Note 1: Diffusion across droplet interface
When dye-labeled macromolecules, such as CF488-BSA or Cy2-RNA, are introduced into the solution surrounding the coacervate droplets, they partition into the protocells by diffusing across the protocell interface.It is evident from the microscopy images that these macromolecules strongly partition into the coacervate matrix.Given this high partitioning, the flux, , of these molecules into the protocells can be modeled as a one-way transport at initial times.The number of molecules crossing the interface of a protocell with a radius  in a Δ time interval can be written as  !" = (4 # )(Δ) .The concentration of these molecules inside the protocells of volume  !" can then be written as Thus, the concentration of partitioned macromolecules inside the protocells at any given time is inversely proportional to their radius, explaining the higher concentration of the guest molecules in the smaller protocells than the larger ones at initial times.

Note 2: NaCl concentration using conductivity measurements
To estimate the amount of counterion ejection from the droplet interface, we measured the conductivity of the water surrounding the stabilized droplets (originally deionized water, but not anymore due to the ejected counterions) and found it to be (45.2± 6.0) µS/cm.Using a calibration curve shown below on the left, we found that this conductivity corresponded to a salt concentration of (0.36 ± 0.036) mM NaCl (mM: mmol/L).
The conductivity of the original equilibrium supernatant was found to be (2295.6± 57.2) µS/cm, measured after centrifuging down the droplets to leave a clear supernatant, which was further filtered using a 0.22 µm filter.The supernatant was found to contain around 2.5 mM of adenosine triphosphate (ATP), which certainly contributed to its conductivity.Using a calibration curve shown above on right, we found that the NaCl concentration in the supernatant is nearly 12.7 mM.However, since there will be some poly(diallyldimethylammonium chloride) (PDDA) in the supernatant in equilibrium with ATP (the concentration of which was not able to be measured due to experimental limitations), it will contribute to the conductivity of the supernatant.Thus, the actual salt concentration in the supernatant will be lower than 12.7 mM.Note that the fitted calibration curve in the plot on the right does not pass through the origin.This is due to the presence of 2.5 mM ATP sodium salt in the standard solutions, the conductivity of which at no added NaCl was found to be 731.2± 15.4 µS/cm, close to the intercept value of the calibration curve.
Below is a summary of the salt amount in the two phases at different stages: Total NaCl in 1 mL original (unmodified) coacervate suspension: 20 mM x 1 mL = 20 µmol (A) To understand the possibility of secondary structures formed by R49 due to the presence of random nucleotides at the 3' end of the sequence that may base pair intra-chain, we performed polyacrylamide gel electrophoresis (PAGE) of this sequence under native (non-denaturing) conditions.The experiments were performed in the absence of any divalent ions, as we did not use any divalent ions in our experiments.It is known that secondary structure(s) of RNA sequence form folded, compact structures that run faster on a native gel due to their compact size (52).We found that the R49 formed a smear, rather than a tight band, in the gel suggesting a range of secondary structures that ran on the gel at different speeds.Compared to R49, Flexizyme ran faster and in a tight band, suggesting most of the molecules had a compact secondary structure, that is smaller than R49.

Figure S2 :
Figure S2: Plots showing the percentage of droplets containing both CF488-BSA and CF640-BSA when the two distinct droplet populations were stabilized and mixed in solutions of different salt concentrations.

Figure S3 :
Figure S3: Plots showing the percentage of droplets containing both CF488-BSA and CF640-BSA when the two distinct droplet populations were stabilized and mixed in solutions of different supernatant fractions.

Figure S4 :
Figure S4: Confocal micrographs showing the stability of PDDA-ATP droplets in low-salt water of varying pH.Droplets containing CF488-BSA and CF640-BSA were first sheared separately in acidic water and were then mixed using a vortex mixer.The acidic water was prepared by adding HCl or NaOH to DI water at different concentrations.Images were taken 30 min after mixing.

Figure S5 :
Figure S5: Measured zeta potential values for unmodified droplets in equilibrium supernatant and stabilized droplets in DI water at different dilutions.1x denotes as-prepared suspensions.A 10x dilution, for e.g., corresponds to a sample where 40 µL of 1x as-prepared suspension was added to 360 µL of corresponding continuous phase.

Figure S6 :
Figure S6: Schematic of different interactions in polyelectrolyte complexes(53).These interactions are broadly of two types: intrinsic and extrinsic ion pairs.Polyanion-polycation pairs are intrinsic ion pairs while polyelectrolyte-counterion pairs are extrinsic ion pairs.An electrostatically crosslinked layer refers to a state where the interfacial layer is dominated by intrinsic ion pairs.

Figure S7 :
Figure S7: (Upper panel) Time series of confocal microscopy images of coacervate droplets in the supernatant (top) and DI water (bottom) before and after photobleaching.The fluorescence signal is from FITC labeled carboxymethyl dextran (ca.900 monomers long) and the dark spot in the center of the droplet at t = 0 s is the photobleached region.Scale bar: 10 µm.(Lower panel) The recovery in fluorescence intensity from the photobleached region.'t' is the time constant of recovery from exponential fits as shown in solid curves.

Figure S8 :
Figure S8: Fluorescence micrographs in a different trial showing the time evolution of Resorufin fluorescence (red channel) in PDDA-ATP coacervate protocells with CF488-GOx (green channel) and CF640-HRP (blue channel) enzymes as cargo.The formation of Resorufin in the HRP protocells is apparent with visibly higher intensity of Resorufin fluorescence in the HRP protocells at short times (t = 0 s and 30 s).

Figure S9 :Figure S10 :
Figure S9: Fraction of RNA transferred from RNA protocells to BSA protocells over time for 6nt and 8-nt long chains.The exchange of RNA of 6-nt length is quite rapid, as evidenced by the significant transfer fraction at t = 0 (when the first image was acquired).Mixing the samples and transferring them to the sample holders for imaging takes ~ 5 min.For each time point, n = 8 samples were analyzed, and error bars represent standard deviations.

Figure S12 :
Figure S12: Fluorescence image of polyacrylamide gel showing size distributions of the Flexizyme and R49 sequences used in this study.~1 µM of 5'-Cy2-labeled RNA was analyzed by polyacrylamide gel electrophoresis (PAGE) under native (non-denaturing) conditions in the absence of any divalent ions.

Table S1 :
Nucleotide sequence of RNA used.Time-lapse showing coalescence of unmodified PDDA-ATP coacervate droplets (i.e. in equilibrium supernatant) using brightfield microscopy.Encircled regions highlight fusion events.Time is in seconds, as shown in the top-right corner.Scale bar: 50 µm, as shown on the bottom-left corner.